Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation.

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.

Estrogen transactivates EGFR via the sphingosine 1-phosphate receptor Edg-3: the role of sphingosine kinase-1.

The transactivation of enhanced growth factor receptor (EGFR) by G protein-coupled receptor (GPCR) ligands is recognized as an important signaling mechanism in the regulation of complex biological processes, such as cancer development. Estrogen (E2), which is a steroid hormone that is intimately implicated in breast cancer, has also been suggested to function via EGFR transactivation. In this study, we demonstrate that E2-induced EGFR transactivation in human breast cancer cells is driven via a novel signaling system controlled by the lipid kinase sphingosine kinase-1 (SphK1). We show that E2 stimulates SphK1 activation and the release of sphingosine 1-phosphate (S1P), by which E2 is capable of activating the S1P receptor Edg-3, resulting in the EGFR transactivation in a matrix metalloprotease-dependent manner. Thus, these findings reveal a key role for SphK1 in the coupling of the signals between three membrane-spanning events induced by E2, S1P, and EGF. They also suggest a new signal transduction model across three individual ligand-receptor systems, i.e., "criss-cross" transactivation.

Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis.

BACKGROUND: Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors. METHODS AND RESULTS: Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1. CONCLUSIONS: We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.

Regulatory network of inflammation downstream of proteinase-activated receptors.

BACKGROUND: Protease-activated receptors (PAR) are present in the urinary bladder, and their expression is altered in response to inflammation. PARs are a unique class of G protein-coupled that carry their own ligands, which remain cryptic until unmasked by proteolytic cleavage. Although the canonical signal transduction pathway downstream of PAR activation and coupling with various G proteins is known and leads to the rapid transcription of genes involved in inflammation, the effect of PAR activation on the downstream transcriptome is unknown. We have shown that intravesical administration of PAR-activating peptides leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P (SP), and antigen was strongly attenuated by PAR1- and to a lesser extent by PAR2-deficiency. RESULTS: Here, cDNA array experiments determined inflammatory genes whose expression is dependent on PAR1 activation. For this purpose, we compared the alteration in gene expression in wild type and PAR1-/- mice induced by classical pro-inflammatory stimuli (LPS, SP, and antigen). 75 transcripts were considered to be dependent on PAR-1 activation and further annotated in silico by Ingenuity Pathways Analysis (IPA) and gene ontology (GO). Selected transcripts were target validated by quantitative PCR (Q-PCR). Among PAR1-dependent transcripts, the following have been implicated in the inflammatory process: b2m, ccl7, cd200, cd63, cdbpd, cfl1, dusp1, fkbp1a, fth1, hspb1, marcksl1, mmp2, myo5a, nfkbia, pax1, plaur, ppia, ptpn1, ptprcap, s100a10, sim2, and tnfaip2. However, a balanced response to signals of injury requires a transient cellular activation of a panel of genes together with inhibitory systems that temper the overwhelming inflammation. In this context, the activation of genes such as dusp1 and nfkbia seems to counter-balance the inflammatory response to PAR activation by limiting prolonged activation of p38 MAPK and increased cytokine production. In contrast, transcripts such as arf6 and dcnt1 that are involved in the mechanism of PAR re-sensitization would tend to perpetuate the inflammatory reaction in response to common pro-inflammatory stimuli. CONCLUSION: The combination of cDNA array results and genomic networks reveals an overriding participation of PAR1 in bladder inflammation, provides a working model for the involvement of downstream signaling, and evokes testable hypotheses regarding the transcriptome downstream of PAR1 activation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestation of cystitis.

Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation.

BACKGROUND: In general, inflammation plays a role in most bladder pathologies and represents a defense reaction to injury that often times is two edged. In particular, bladder neurogenic inflammation involves the participation of mast cells and sensory nerves. Increased mast cell numbers and tryptase release represent one of the prevalent etiologic theories for interstitial cystitis and other urinary bladder inflammatory conditions. The activity of mast cell-derived tryptase as well as thrombin is significantly increased during inflammation. Those enzymes activate specific G-protein coupled proteinase-activated receptors (PAR)s. Four PARs have been cloned so far, and not only are all four receptors highly expressed in different cell types of the mouse urinary bladder, but their expression is altered during experimental bladder inflammation. We hypothesize that PARs may link mast cell-derived proteases to bladder inflammation and, therefore, play a fundamental role in the pathogenesis of cystitis. RESULTS: Here, we demonstrate that in addition to the mouse urinary bladder, all four PA receptors are also expressed in the J82 human urothelial cell line. Intravesical administration of PAR-activating peptides in mice leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS), substance P, and antigen was strongly attenuated by PAR1-, and to a lesser extent, by PAR2-deficiency. CONCLUSION: Our results reveal an overriding participation of PAR1 in bladder inflammation, provide a working model for the involvement of downstream signaling, and evoke testable hypotheses regarding the role of PARs in bladder inflammation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestations of cystitis.

Novel indolylindazolylmaleimides as inhibitors of protein kinase C-beta: synthesis, biological activity, and cardiovascular safety.

Novel indolylindazolylmaleimides were synthesized and examined for kinase inhibition. We identified low-nanomolar inhibitors of PKC-beta with good to excellent selectivity vs other PKC isozymes and GSK-3beta. In a cell-based functional assay, 8f and 8i effectively blocked IL-8 release induced by PKC-betaII (IC(50) = 20-25 nM). In cardiovascular safety assessment, representative lead compounds bound to the hERG channel with high affinity, potently inhibited ion current in a patch-clamp experiment, and caused a dose-dependent increase of QT(c) in guinea pigs.

Extracellular mediators in atherosclerosis and thrombosis: lessons from thrombin receptor knockout mice.

It is well appreciated that thrombin as well as other proteases can act as signaling molecules that specifically regulate cells by cleaving and activating members of a novel class of protease-activated receptors (PARs). The utility of gene knockout strategies to define and better comprehend the physiological role of specific proteins is perhaps best exemplified in the field of thrombin receptors. The development of PAR knockout mice has provided the unique opportunity to identify and characterize new members of this novel family of GPCRs, evaluate the interaction of PARs jointly expressed in common cells and tissues, and better understand the role of PARs in thrombosis, restenosis, vascular remodeling, angiogenesis, and inflammation. Presently, 4 members of the PAR family have been cloned and identified. In this review, we examine experimental evidence gleaned from PAR-/- mouse models as well as how the use of PAR-/- mice has provided insights toward understanding the physiological role of thrombin in cells of the vascular system and vascular pathology.

Mer receptor tyrosine kinase signaling participates in platelet function.

OBJECTIVE: Recently, mice made deficient in growth arrest-specific gene 6 product (Gas6) or in which Gas6 gene expression was inhibited were shown to have platelet dysfunction and to be less susceptible to thrombosis. The aim of this study was to define and characterize the relevant Gas6 receptor or receptors involved in platelet function. METHODS AND RESULTS: Using RT-PCR and Western blot analysis we found that mer was the predominantly expressed subtype in mouse and human platelets, whereas axl and rse were not detected. We generated mer-deficient mice by targeted disruption of the mer receptor gene. Platelets derived from mer-deficient mice had decreased platelet aggregation in responses to low concentrations of collagen, U46619, and PAR4 thrombin receptor agonist peptide in vitro. However, the response to ADP was not different from wild-type platelets. Knockout of the mer gene protected mice from collagen/epinephrine-induced pulmonary thromoembolism and inhibited ferric chloride-induced thrombosis in vivo. Tail bleeding times, coagulation parameters, and peripheral blood cell counts in mer-deficient mice were similar to wild-type mice. CONCLUSIONS: Our data provide the first evidence that mer, presumably through activation by its ligand Gas6, participates in regulation of platelet function in vitro and platelet-dependent thrombosis in vivo.

High-throughput siRNA-based functional target validation.

The drug discovery process pursued by major pharmaceutical companies for many years starts with target identification followed by high-throughput screening (HTS) with the goal of identifying lead compounds. To accomplish this goal, significant resources are invested into automation of the screening process or HTS. Robotic systems capable of handling thousands of data points per day are implemented across the pharmaceutical sector. Many of these systems are amenable to handling cell-based screening protocols as well. On the other hand, as companies strive to develop innovative products based on novel mechanisms of action(s), one of the current bottlenecks of the industry is the target validation process. Traditionally, bioinformatics and HTS groups operate separately at different stages of the drug discovery process. The authors describe the convergence and integration of HTS and bioinformatics to perform high-throughput target functional identification and validation. As an example of this approach, they initiated a project with a functional cell-based screen for a biological process of interest using libraries of small interfering RNA (siRNA) molecules. In this protocol, siRNAs function as potent gene-specific inhibitors. siRNA-mediated knockdown of the target genes is confirmed by TaqMan analysis, and genes with impacts on biological functions of interest are selected for further analysis. Once the genes are confirmed and further validated, they may be used for HTS to yield lead compounds.

Functional informatics: convergence and integration of automation and bioinformatics.

The biopharmaceutical industry is currently being presented with opportunities to improve research and business efficiency via automation and the integration of various systems. In the examples discussed, industrial high-throughput screening systems are integrated with functional tools and bioinformatics to facilitate target and biomarker identification and validation. These integrative functional approaches generate value-added opportunities by leveraging available automation and information technologies into new applications that are broadly applicable to different types of projects, and by improving the overall research and development and business efficiency via the integration of various systems.

Therapeutic potential of protease-activated receptor-1 antagonists.

The serine protease thrombin (EC 3.4.21.5) is central to the maintenance of haemostatic balance through its coagulant, anticoagulant and platelet activating properties. In addition, this enzyme affects numerous cellular responses in a wide variety of cells, such as cell proliferation, cytokine and growth factor release, lipid metabolism and tissue remodelling. A family of G-protein-coupled protease-activated receptors (PARs) mediates these cellular actions of thrombin. While thrombin can activate three of the four PAR family members, PAR-1 represents the primary thrombin-responsive receptor in human cells. The expression of PAR-1 in platelets, the vasculature and myocardium, in cells within atherosclerotic plaque and tissues after vascular injury, indicates that this receptor plays an important role during the response to tissue injury and associated inflammatory processes. With the development of PAR-deficient mice and small-molecule antagonists, it is now clear that intervening in processes mediated by PAR-1 presents a new approach to treating a variety of disorders dependent on thrombin generation, including thrombosis and restenosis. The full potential of PAR-1 antagonists has yet to be realised, but the promise of novel therapeutics that modulate receptor function rather than thrombin's proteolytic activity, provides an alternative and, perhaps, more desirable means to dampen the pathological effects of thrombin.

Discovery of potent peptide-mimetic antagonists for the human thrombin receptor, protease-activated receptor-1 (PAR-1).

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g. platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.

Increased local expression of coagulation factor X contributes to the fibrotic response in human and murine lung injury.

Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Coagulation zymogens are considered to be largely derived from the circulation and locally activated in response to tissue injury and microvascular leak. Here we report that expression of coagulation factor X (FX) is locally increased in human and murine fibrotic lung tissue, with marked immunostaining associated with bronchial and alveolar epithelia. FXa was a potent inducer of the myofibroblast differentiation program in cultured primary human adult lung fibroblasts via TGF-beta activation that was mediated by proteinase-activated receptor-1 (PAR1) and integrin alphavbeta5. PAR1, alphavbeta5, and alpha-SMA colocalized to fibrotic foci in lung biopsy specimens from individuals with idiopathic pulmonary fibrosis. Moreover, we demonstrated a causal link between FXa and fibrosis development by showing that a direct FXa inhibitor attenuated bleomycin-induced pulmonary fibrosis in mice. These data support what we believe to be a novel pathogenetic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally expressed and drives the fibrotic response to lung injury. These findings herald a shift in our understanding of the origins of excessive procoagulant activity and place PAR1 central to the cross-talk between local procoagulant signaling and tissue remodeling.

Novel bis(indolyl)maleimide pyridinophanes that are potent, selective inhibitors of glycogen synthase kinase-3.

Novel bis(indolyl)maleimide pyridinophanes 3, 9a, 9b, 10a, 10b, and 11 were prepared by cobalt-mediated [2+2+2] cycloaddition of an appropriate alpha,omega-diyne with an N,N-dialkylcyanamide. These macrocyclic heterophanes were found to be potent, selective inhibitors of glycogen synthase kinase-3beta. An X-ray structure of a co-crystal of GSK-3beta and 3 (IC(50)=3nM) depicts the hydrogen bonding and hydrophobic interactions in the ATP-binding pocket of this serine/threonine protein kinase.

Integrated expressional analysis: application to the drug discovery process.

Microarray technology enables high-throughput testing of gene expression to investigate various neuroscience related questions. This in turn creates a demand for scalable methods to confirm microarray results and the opportunity to use this information to discover and test novel pathways and therapeutic applications. Discovery of new central nervous system (CNS) treatments requires a comprehensive understanding of multiple aspects including the biology of a target, the pathophysiology of a disease/disorder, and the selection of successful lead compounds as well as efficient biomarker and drug disposition strategies such as absorption (how a drug is absorbed), distribution (how a drug spreads through an organism), metabolism (chemical conversion of a drug, if any, and into which substances), and elimination (how is a drug eliminated) (ADME). Understanding of the toxicity is also of paramount importance. These approaches, in turn, require novel high-content integrative assay technologies that provide thorough information about changes in cell biology. To increase efficiency of profiling, characterization, and validation, we established a new screening strategy that combines high-content image-based testing on Array Scan (Cellomics) with a confocal system and the multiplexed TaqMan RT-PCR method for quantitative mRNA expression analysis. This approach could serve as an interface between high-throughput microarray testing and specific application of markers discovered in the course of a microarray experiment. Markers could pinpoint activation or inhibition of a molecular pathway related, for instance, to neuronal viability. We demonstrate the successful testing of the same cell population in an image-based translocational assay followed by poly(A) mRNA capture and multiplexed single tube RT-PCR. In addition, Ciphergen ProteinChip analysis can be performed on the supernatant, thus allowing significant complementarity in the data output and interpretation by also including the capture and initial analysis of proteins in the integrative approach presented. We have determined various conditions including the number of cells, RT and PCR optimization, which are necessary for successful detection and consequent assay integration. We also show the successful convergence of various different approaches and multiplexing of different targets within a single real-time PCR tube. This novel integrative technological approach has utility for CNS drug discovery, target and biomarker identification, selection and characterization as well as for the study of toxicity- and adverse event-associated molecular mechanisms.

A novel, potent dual inhibitor of the leukocyte proteases cathepsin G and chymase: molecular mechanisms and anti-inflammatory activity in vivo.

Certain leukocytes release serine proteases that sustain inflammatory processes and cause disease conditions, such as asthma and chronic obstructive pulmonary disease. We identified beta-ketophosphonate 1 (JNJ-10311795; RWJ-355871) as a novel, potent dual inhibitor of neutrophil cathepsin G (K(i) = 38 nm) and mast cell chymase (K(i) = 2.3 nm). The x-ray crystal structures of 1 complexed with human cathepsin G (1.85 A) and human chymase (1.90 A) reveal the molecular basis of the dual inhibition. Ligand 1 occupies the S(1) and S(2) subsites of cathepsin G and chymase similarly, with the 2-naphthyl in S(1), the 1-naphthyl in S(2), and the phosphonate group in a complex network of hydrogen bonds. Surprisingly, however, the carboxamido-N-(naphthalene-2-carboxyl)piperidine group is found to bind in two distinct conformations. In cathepsin G, this group occupies the hydrophobic S(3)/S(4) subsites, whereas in chymase, it does not; rather, it folds onto the 1-naphthyl group of the inhibitor itself. Compound 1 exhibited noteworthy anti-inflammatory activity in rats for glycogen-induced peritonitis and lipopolysaccharide-induced airway inflammation. In addition to a marked reduction in neutrophil influx, 1 reversed increases in inflammatory mediators interleukin-1alpha, interleukin-1beta, tissue necrosis factor-alpha, and monocyte chemotactic protein-1 in the glycogen model and reversed increases in airway nitric oxide levels in the lipopolysaccharide model. These findings demonstrate that it is possible to inhibit both cathepsin G and chymase with a single molecule and suggest an exciting opportunity in the treatment of asthma and chronic obstructive pulmonary disease.

Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4.

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.

Characterization of thrombin-induced leukocyte rolling and adherence: a potential proinflammatory role for proteinase-activated receptor-4.

It is commonly accepted that thrombin exerts its proinflammatory properties through the activation of proteinase-activated receptor (PAR)-1, although two other thrombin receptors have been discovered: PAR-3 and PAR-4. In this study, we have investigated the mechanisms and the receptors involved in thrombin-induced leukocyte/endothelial cell interactions by using selective agonists and antagonists of thrombin receptors in an in vivo intravital microscopy system. Topical addition of selective PAR-1 agonists to rat mesenteric venules failed to reproduce the increased leukocyte rolling and adhesion observed after thrombin topical addition. When added together with the selective PAR-1 antagonist RWJ-56110, thrombin was still able to provoke increased leukocyte rolling and adherence. The thrombin-induced leukocyte rolling and adherence was not affected by pretreatment of rats with an anti-platelet serum. Selective PAR-4-activating peptide was able to reproduce the effects of thrombin on leukocyte rolling and adhesion. Intraperitoneal injection of PAR-4-activating peptide also caused a significant increase in leukocyte migration into the peritoneal cavity. In rat tissues, PAR-4 expression was detected both on endothelium and isolated leukocytes. Taken together, these results showed that in rat mesenteric venules, thrombin exerts proinflammatory properties inducing leukocyte rolling and adherence, by a mechanism independent of PAR-1 activation or platelet activation. However, PAR-4 activation either on endothelial cells or on leukocytes might be responsible for the thrombin-induced effects. These findings suggest that PAR-4 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence and recruitment, and that in addition to PAR-1, PAR-4 could be involved in proinflammatory properties of thrombin.

RWJ-58259: a selective antagonist of protease activated receptor-1.

Protease activated receptor-1 (PAR-1) is a key mediator of the cellular actions of alpha-thrombin. Thus, antagonism of this unique G-protein coupled receptor with a small molecule represents a means of selectively inhibiting thrombin's cellular actions without inhibiting its proteolytic activity. RWJ-58259 (alphaS)-N-[(1S)-3-amino-1-[[(phenylmethyl)- amino]carbonyl]propyl]-alpha-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide) is a potent and selective inhibitor of PAR-1 identified as part of a synthetic chemistry program based upon a de novo design approach. RWJ-58259 inhibited thrombin-induced platelet aggregation in human platelets with an IC50 of 0.37 microM without inhibiting thrombin's proteolytic activity or aggregation induced by other agonists. RWJ-58259 was not effective in guinea pig models of thrombosis. This reflected the presence of a second thrombin-sensitive receptor system in guinea pigs (PAR-3/4) and the selectivity of RWJ-58259 for PAR-1. However, RWJ-58259 was effective in a non-human primate model of thrombosis. Because human platelets have a PAR expression profile similar to the non-human primate, PAR-1 antagonism has the potential to be antithrombotic in humans. RWJ-58259 also inhibited thrombin-induced intracellular calcium signaling and proliferation in rat vascular smooth muscle cells. Perivascular application of RWJ-58259 in vivo significantly inhibited arterial injury-induced stenosis in a rat model of balloon angioplasty. These preclinical results suggest a potential clinical utility of RWJ-58259 for treatment of thrombotic disorders and vascular injury associated with acute coronary interventions and atherosclerosis. Given the potential role of PAR-1 in thrombin's actions in other cell types and disease states, RWJ-58259 provides a means for assessing additional clinical utilities of PAR-1 antagonism in disease conditions such as inflammation, cancer and neurodegeneration.

An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes.

Sphingosine kinase catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, a lipid signaling molecule implicated in roles in a diverse range of mammalian cell processes through its action as both a ligand for G-protein-coupled cell-surface receptors and an apparent intracellular second messenger. This paper describes a rapid, sensitive, and reproducible assay for sphingosine kinase activity using biotinylated sphingosine (biotinyl-Sph) as a substrate and capturing the phosphorylated product with streptavidin-coated membranes. We have shown that both human sphingosine kinase 1 and 2 (hSK1 and hSK2) can efficiently phosphorylate biotinyl-Sph, with K(m) values similar to those of sphingosine. The assay utilizing this substrate has high sensitivity for hSK1 and hSK2, with detection limits in the low-femtomole range for both purified recombinant enzymes. Importantly, we have also demonstrated the capacity of this assay to measure endogenous sphingosine kinase activity in crude cell extracts and to follow changes in this activity following sphingosine kinase activation. Together, these results demonstrate the potential utility of this assay in both cell-based analysis of sphingosine kinase signaling pathways and high-throughput screens for agents affecting sphingosine kinase activity in vitro.